PROJECT SUMMARY The global spread of arthropod borne dengue virus (DENV, serotypes 1-4) is a major global public health challenge. World health organizations are calling for scientific communities to respond to this emerging threat with vaccines, therapeutics and diagnostics. However, existing gaps in fundamental knowledge about DENV immunity, specifically how type-specific immunity to each serotype develops and is sustained, makes rational vaccine development particularly difficult. The dominant immunity model comes from human challenge studies on DENV (1910?s-1940?s) and posits that first infection induces neutralizing antibodies (NAbs) that provide life- long sterilizing immunity to repeat infections by the same (homotypic) serotype virus. However, recent studies from DENV endemic regions provide evidence of homotypic re-infection and natural immune boosting over time and our preliminary data from a non-endemic, Portland, Oregon resident DENV immune cohort show type-specific DENV NAbs decay over time, calling into question sterilizing immunity and leading us to hypothesize that natural DENV protective immunity may in fact rely on intermittent boosting, as would occur in endemic settings. To test this hypothesis, we propose to directly compare DENV immunity in a regularly re- exposed endemic cohort with immunity in an un-boosted, non-endemic cohort of DENV immunes. This is a controversial and novel hypothesis that will call for substantial supporting evidence, hence we will evaluate two highly relevant and related markers of boosting: NAbs (Aim 1) and memory B-cells (MBCs) (Aim 2) in the two cohorts. We expect to find that NAbs and MBCs decay at a significantly greater rate in the non-endemic setting, supporting the hypothesis that boosting plays a critical role in natural immunity for these viruses, with implications for future vaccine development. In Aim 1 we will use both longitudinal and prospectively collected samples to evaluate potency and breadth of DENV immune serum NAb in both cohorts and compare the rates at which NAb titers decay. We hypothesize that DENV NAb titers will decay more rapidly in the non-endemic cohort compared to the endemic cohort. In Aim 2 we use the same approach detailed in Aim 1, to evaluate the frequency of DENV specific MBCs in the endemic and non-endemic cohorts. We hypothesize that frequencies will decay more rapidly in the non-endemic cohort. Because MBCs are functionally linked to NAbs, quantifying virus specific MBCs tests a mechanistic connection between repeat infection and NAb titer boosting. Our proposed work will comprehensively assess the validity of sterilizing immunity using two highly relevant and mechanistically linked determinants (NAb and MBC). Irrespective of specific results, the knowledge obtained from the proposed work will be essential to effective and rational DENV vaccine development.